Anti-CRISPR (Acr) proteins are phage-borne inhibitors of the CRISPR-Cas immune system in archaea and bacteria. AcrIIC2 from prophages of Neisseria meningitidis disables the nuclease activity of type II-C Cas9, such that dimeric AcrIIC2 associates with the bridge helix (BH) region of Cas9 to compete with guide RNA loading. AcrIIC2 in solution readily assembles into oligomers of variable lengths, but the oligomeric states are not clearly understood. In this study, we investigated the dynamic assembly of AcrIIC2 oligomers, and identified key interactions underlying the self-association. We report that AcrIIC2 dimers associate into heterogeneous high-order oligomers with the equilibrium dissociation constant K_D ∼8 μM. Oligomerization is driven by electrostatic interactions between charged residues, and rational mutagenesis produces a stable AcrIIC2 dimer with intact Cas9 binding. Remarkably, the BH peptide of Cas9 is unstructured in solution, and undergoes a coil-to-helix transition upon AcrIIC2 binding, revealing a unique folding-upon-binding mechanism for Acr recognition.

中文翻译：

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Anti-CRISPR AcrIIC2自组装和靶标结合的结构研究

抗 CRISPR (Acr) 蛋白是古细菌和细菌中 CRISPR-Cas 免疫系统的噬菌体抑制剂。来自脑膜炎奈瑟菌原噬菌体的 AcrIIC2使 II-C 型 Cas9 的核酸酶活性失效，使得二聚体 AcrIIC2 与 Cas9 的桥螺旋 (BH) 区域结合，与引导 RNA 加载竞争。溶液中的 AcrIIC2 很容易组装成可变长度的低聚物，但低聚物状态尚不清楚。在这项研究中，我们研究了 AcrIIC2 寡聚体的动态组装，并确定了自关联背后的关键相互作用。我们报告 AcrIIC2 二聚体与平衡解离常数K _D结合成异质高阶低聚物～8 μM。寡聚化由带电残基之间的静电相互作用驱动，合理的诱变产生稳定的 AcrIIC2 二聚体，具有完整的 Cas9 结合。值得注意的是，Cas9 的 BH 肽在溶液中是非结构化的，并且在 AcrIIC2 结合后经历了线圈到螺旋的转变，揭示了 Acr 识别的独特的结合折叠机制。